While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein–encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.

Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.Human erythropoietin (EPO) is a glycoprotein hormone that plays a major role in regulating the level of circulating erythrocytes (1) by stimulating the maturation of late erythroid progenitor cells into proerythroblasts (2). In the normal human adult, EPO is produced in the kidney (3). EPO was first purified in a small amount from urine of aplastic anemia patients (4). Several recombinant human EPOs produced in mammalian (5-9) and nonmammalian cells (10) have recently become available, but their biological activities differ from cell to cell. Since the structures of their polypeptide moieties are the same, such variation in activity was suspected as being due to the differences in their carbohydrate moieties. The sugar chain structures of urinary human EPO and recombinant human EPO produced in Chinese hamster ovary (CHO) cells were determined independently by us (11) and by Sasaki et al. (12). Both urinary and recombinant human EPOs contain -40% carbohydrate in the form of three N-linked and one O-linked oligosaccharide chain. Important roles of the carbohydrate moiety in the solubility (13, 14), biosynthesis (14), and biological activity of EPO (9, 15) have been reported. Desialylation of EPO caused complete loss of its hormonal activity in vivo (15, 16) as the asialo-EPO was trapped in the liver (17) by the hepatic asialoglycoprotein binding protein (18) and was rapidly cleared from the circulation. Galactose oxidase treatment of asialo-EPO restored part of the biological activity (16). These results suggested that EPO possesses full biological activity only when it is sufficiently sialylated to avoid clearance by the hepatic asialoglycoprotein binding protein. During the course of study of the productivity of EPO in several recombinant CHO cell lines, we found a unique cell line, B8-300, which produced ...

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